Telomere integrity is important for mammalian cell survival and proliferation. Telomere attrition or exposed chromosomal ends could result in genome instability, DNA damage response, and ultimately cancer. Mutations in telomerase subunits or telomere regulators have been linked to premature aging and cancer. Understanding the cellular mechanisms of telomere maintenance may therefore help in developing therapies for diseases including cancer. The human 3'overhang binding protein POT1 helps to protect the telomere ssDNA and regulate telomerase access. Recently, the lab identified a new telomere associated protein TPP1/PTOP (also known as PIP1/TINT1). We found it to directly interact with POT1 and the telomerase. Furthermore, TPP1 forms the human telosome, along with POT1, TRF1, TRF2, RAP1, and TIN2. Our findings suggest that TPP1 controls telomere length by regulating telomere recruitment of POT1 and telomerase. The long-term goal is to study how dysregulation of telosome components may lead to telomere dysfunction and cancer development. The overall objective of this proposal is to understand the function of TPP1 in telomere protection and hematopoietic cancer. We hypothesize that TPP1 may heterodimerize with POT1 to protect telomere ends, and control telomerase access and activity. Specific Aim 1. To determine the molecular mechanism of TPP1-mediated telomere end protection and length control. (1) The dynamic assembly, protein stability, and localization of the POT1-TPP1 complex in human cells will be investigated. We will determine protein modifications such as ubiquitination, and the cell cycle regulated localization and organization of the POT1-TPP1 complex. (2) The role of TPP1 in telomere end protection, cell survival, and cell cycle checkpoint activation will be determined through knockout, RNAi, and dominant negative expression. Specific Aim 2: To elucidate the role of TPP1 in controlling telomerase activity. How TPP1 interacts with the telomerase and controls telomerase activity, regulates cell cycle-dependent telomerase access to telomeres will be studied. Specific Aim 3: To investigate the consequences of telomere dysfunction in development and cancer. Chimeric mice whose hematopoietic systems are reconstituted with cells expressing dominant-negative TPP1 and TPP1 shRNA will be generated, and used to determine how TPP1 affects hematopoietic survival and development of hematopoietic cancers. The studies described in this proposal should help to delineate the signaling mechanisms by which TPP1 regulates telomere maintenance in vivo, and provide insight into the regulation and mechanisms of TPP1 in development and telomere protection, especially during hematopoiesis. This work may provide a more refined picture about the complex signaling networks that are employed by the cells to control genome stability. Furthermore, valuable targets for mechanism-driven design of cancer and aging therapeutics may be identified through this project.